The red cell mass is regulated by the erythropoietic cytokine, erythropoietin (Epo), which is produced in the kidney and liver in response to hypoxia. In Hep3B cells exposure to 1 percent O2 results in a 50-100 fold increase in steady-state levels of Epo mRNA, due largely to enhanced transcription. Transcriptional regulation appears to depend on the interplay of Hypoxia Inducible Factor 1 (HIF-1), Hepatic Nuclear Receptor 4 (HNF-4), and p300. HIF-1 and HNF-4 are transcription factors that bind to a 3' Epo enhancer, while p300 interacts with the alpha subunit of HIF-1 to enhance transcription. In Specific Aim 1, experiments are designed to identify the specific interactions among HIF-1, HNF-4 and p300. Identification of protein-protein interactions in vivo by immunoprecipitation will confirm the preliminary results obtained from several independent studies. Direct protein-protein interactions will be verified in vitro by GST pull-down assays. The functional role of these interactions will be examined in vivo by using a mammlian two hybrid system. Specific Aim 2 entails using a retroviral gene transfer system to study the biological role of HNF-4 in tissue-specific hypoxic induction of Epo gene. HNF-4 will be transduced into variety of cells to test if it can force expression of the endogenous Epo gene. In addition, hepatic nuclear factor 1 and nuclear receptors EAR3/COUP-TF1 and TR2, known to counteract HNF-4 activity, will be used to investigate their impact on Epo gene expression. These studies should advance our understanding of the molecular basis of Epo gene induction and may provide new insights into more general mechanisms by which cells and organisms respond to hypoxic stress.